Life begins with the fusion of an oocyte and a sperm cell. These two cells each carry half the genetic information required for life, and unite to form a single cell that contains a complete set of DNA. This cell embarks on a remarkable process, transforming into a complex organi
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Life begins with the fusion of an oocyte and a sperm cell. These two cells each carry half the genetic information required for life, and unite to form a single cell that contains a complete set of DNA. This cell embarks on a remarkable process, transforming into a complex organism made up of trillions of cells. At the core of this transformation lies the process of cellular duplication, in which one cell divides to form two, then four, and so on, exponentially increasing in number. Through countless rounds of division, these cells differentiate to form the tissues, organs, and systems that define the human body. Despite the incredible diversity in their functions - from neurons transmitting signals to muscle cells contracting - the DNA within each cell remains identical to the fertilized egg cell. The precise duplication is crucial, as the DNA carries the instructions for building and maintaining the body. Yet, the scale of this task is staggering: ensuring that billions of cells inherit the exact same genetic material without error over the course of development and throughout life. This raises a fundamental question: how is DNA evenly distributed during each cell division?
At the heart of this challenge lies the structure of DNA, organized into 46 long, thread-like molecules each carrying a specific set of genetic instructions. These threads can be thought of as 46 strands of spaghetti tangled together in a bowl. Before a cell divides, it replicates the DNA to double the number of strands from 46 to 92. This replication event enables the two new cells to inherit one complete set of 46 chromosomes each. However, errors in this distribution can have severe consequences, such as missing or extra chromosomes in the new cells. To prevent such errors, cells have developed sophisticated mechanisms to tether replicated DNA strands together, allowing the cell to “remember” which strands must be pulled to opposite sides during division. Central to this process is the cohesin protein complex, which functions as a molecular ring that encircles the duplicated DNAs from the moment of DNA replication onwards. These cohesin rings tightly hold together the DNAs until the cell is ready to divide. At that moment, the cohesin rings are removed and the replicated DNAs move to opposite sides of the cell. Cohesin hereby ensures an equal and accurate distribution of DNA to the two new cells.
Key to the tethering of the two replicated DNAs is the capture of these DNAs into the cohesin complex. In chapter 4, we reveal that two proteins (CTCF and MCM3) promote this capture. Importantly, we show that these proteins each do so by binding to the same region on the cohesin complex. We refer to this region on cohesin as the Conserved Essential Surface (CES), that we pronounce as “Chess” (hence the cover of this thesis). We show that CTCF and MCM3 are not the only proteins that interact with the CES. Chapter 6 reveals that WAPL also binds with this region to promote the opening of cohesin rings and their release from DNA. In chapter 3 we show that the protein Shugoshin (SGO1) interacts with the CES to counteract WAPL-driven DNA release activity, thereby safeguarding the cohesin rings that hold the replicated DNAs together.
Cohesin not only holds together the two replicated DNAs, but also shapes DNA threads by building and enlarging DNA loops. These loops bring distant regions of DNA together, but the exact mechanism by which cohesin builds these loops remains unclear. In chapter 8, we integrate recent literature to propose a model for the enlargement of DNA loops. In chapter 2, we reveal that CTCF not only interacts with the CES to control the complexes that hold together replicated DNAs, but also to control the cohesin complexes involved in loop formation. This thesis thus uncovers a novel mechanism of cohesin regulation, in which multiple proteins compete for binding to a single surface on cohesin, controlling cohesin irrespective of whether it is building loops or holding together replicated DNAs. In chapter 9, we discuss how these findings contribute to our current understanding of cohesin’s ability to shape DNAs. However, many questions remain. For instance, what mechanistically happens when these regulators bind the CES? And how different are the cohesin complexes that build loops or tether the replicated DNAs really? In short, there is still much to uncover.